US5055412AExpiredUtility

Factor sensitive reagent for testing of blood coagulation containing ellagic acid and divalent metal ions and method of making the same

81
Individually held — no corporate assignee on recordPriority: Mar 21, 1989Filed: Mar 21, 1989Granted: Oct 8, 1991
Est. expiryMar 21, 2009(expired)· nominal 20-yr term from priority
Inventors:Gary J. Proksch
G01N 33/86G01N 2405/04
81
PatentIndex Score
50
Cited by
7
References
53
Claims

Abstract

The present invention involves a partial thromboplastin time test reagent which is factor sensitive depending upon the selected reagent constituents. A method for peparing such reagent is also disclosed. The present invention further involves a procoagulant precursor reagent and a method of making it. The factor sensitive reagent is of exceptional stability enabling more precise partial prothrombin time measurements. The reagents contain ellagic acid or an ellagic acid salt and divaent metal ions in a specific molar ratio relative to the ellagic acid or ellagic acid salt.

Claims

exact text as granted — not AI-modified
What is claimed is: 
     
       1. A stable factor sensitive reagent for measuring partial thromboplastin time comprising: ellagic acid or a ellagic acid salt present at a predetermined molar concentration;   a divalent metal ion, said divalent metal ion present at a molar ratio between 3 and 30 to said ellagic acid or ellagic acid salt; and,   a cephalin.   
     
     
       2. The reagent of claim 1 wherein the molar ratio of said divalent metal ion to said ellagic acid or ellagic acid salt is between about 4 and about 10. 
     
     
       3. The reagent of claim 2 wherein said ellagic acid or ellagic acid salt is sodium ellagate. 
     
     
       4. The reagent of claim 2 wherein said cephalin is soybean cephalin. 
     
     
       5. The reagent of claim 2 wherein said divalent metal ion is selected from the group consisting of: magnesium;   calcium;   copper;   cobalt;   iron;   lead;   manganese;   strontium; and,   zinc.   
     
     
       6. The reagent of claim 5 wherein said divalent metal ion is manganese. 
     
     
       7. The reagent of claim 5 wherein said divalent metal ion is magnesium. 
     
     
       8. The reagent of claim 1 wherein the predetermined molar concentration is approximately 0.1 millimolar. 
     
     
       9. The reagent of claim 8 and further wherein the divalent metal ion concentration is between 0.7 millimolar and 0.3 millimolar. 
     
     
       10. A stable factor sensitive reagent for measuring partial thromboplastin time comprising: ellagic acid or an ellagic acid salt present at a predetermined molar concentration;   divalent metal ions, said divalent metal ions present at a molar ratio between 3 and 30 to said ellagic acid or ellagic acid salt; and,   a cephalin.   
     
     
       11. The reagent of claim 10 wherein the molar ratio of said divalent metal ions to said ellagic acid or ellagic acid salt is between about 4 and about 10. 
     
     
       12. The reagent of claim 11 wherein said ellagic acid or ellagic acid salt is sodium ellagate. 
     
     
       13. The reagent of claim 11 wherein said divalent metal ions are selected from the group consisting of: magnesium;   calcium;   copper;   cobalt;   iron;   lead;   manganese;   strontium; and   zinc.   
     
     
       14. The reagent of claim 13 wherein said divalent metal ions include manganese. 
     
     
       15. The reagent of claim 13 wherein said divalent metal ions include magnesium. 
     
     
       16. The reagent of claim 10 wherein the predetermined molar concentration is approximately 0.1 millimolar. 
     
     
       17. The reagent of claim 16 and further wherein the concentration of divalent metal ions is between 0.7 millimolar and 0.3 millimolar. 
     
     
       18. The reagent of claim 10 wherein said cephalin is soybean cephalin. 
     
     
       19. A stable reagent capable of forming a procoagulation reagent upon exposure to a source of cephalin consisting essentially of: ellagic acid or an ellagic acid salt present at a predetermined molar concentration; and,   a divalent metal ion, said divalent metal ion present at a molar ratio of 3 or less, but greater than 0.1, to said ellagic acid or ellagic acid salt.   
     
     
       20. The reagent of claim 19 wherein the molar ratio of said divalent metal ion to said ellagic acid or ellagic acid salt is 2 or less but greater than 0.5. 
     
     
       21. The reagent of claim 20 wherein said divalent metal ion is selected from the group consisting of: magnesium;   calcium;   copper;   cobalt;   iron;   lead;   manganese;   strontium; and,   zinc.   
     
     
       22. The reagent of claim 20 wherein the divalent metal ion is of low relative toxicity. 
     
     
       23. The reagent of claim 19 wherein said ellagic acid or ellagic acid salt is sodium ellagate. 
     
     
       24. The reagent of claim 19 wherein the predetermined molar concentration of ellagic acid or ellagic acid salt is approximately 0.1 millimolar. 
     
     
       25. The reagent of claim 24 and further wherein the divalent metal ion concentration is approximately 0.05 millimolar. 
     
     
       26. The reagent of claim 19 wherein said divalent metal ion is selected from the group consisting of: magnesium;   calcium;   copper;   cobalt;   iron;   lead;   manganese;   strontium; and,   zinc.   
     
     
       27. The reagent of claim 26 wherein the divalent metal ion is magnesium. 
     
     
       28. A stable reagent capable of forming a procoagulation reagent upon exposure to a source of cephalin consisting essentially of: ellagic acid or an ellagic acid salt present at a predetermined molar concentration; and,   divalent metal ions, said divalent metal ions present at a molar ratio of 3 or less, but greater than 0.1, to said ellagic acid or ellagic acid salt.   
     
     
       29. The reagent of claim 28 wherein the molar ratio of said divalent metal ions to said ellagic acid or ellagic acid salt is 2 or less but greater than 0.5. 
     
     
       30. The reagent of claim 29 wherein said ellagic acid or ellagic acid salt is sodium ellagate. 
     
     
       31. The reagent of claim 29 wherein said divalent metal ions are selected from the group consisting of: magnesium;   calcium;   copper;   cobalt;   iron;   lead;   manganese;   strontium; and,   zinc.   
     
     
       32. The reagent of claim 28 wherein the predetermined molar concentration of ellagic acid or ellagic acid salt is approximately 0.1 millimolar. 
     
     
       33. The reagent of claim 32 and further wherein the concentration of divalent metal ions is approximately 0.05 millimolar. 
     
     
       34. The reagent of claim 28 wherein said divalent metal ions are selected from the group consisting of: magnesium;   calcium;   copper;   cobalt;   iron;   lead;   manganese;   strontium; and,   zinc.   
     
     
       35. The reagent of claim 34 wherein the divalent metal ions include magnesium. 
     
     
       36. The reagent of claim 34 wherein the divalent metal ions are of low relative toxicity. 
     
     
       37. A method of making a stable factor sensitive reagent for measuring partial thromboplastin time comprising the sequential steps of: first, dissolving a known weight of an ellagic acid or an ellagic acid salt in an aqueous solution;   second, adding a cephalin to the above resulting solution;   third, adjusting the concentration of divalant metal ions of said solution to achieve a divalent metal ion molar ratio between 3 and 30 relative to the ellagic acid or ellagic acid salt of said solution; and,   fourth, adjusting the pH of the resulting ellagic acid solution to approximately 7.5.   
     
     
       38. The method of claim 37 and further comprising assaying one or more of said cephalin, said ellagic acid or ellagic acid salt, or said aqueous solution prior to said adjusting the divalent metal ion concentration. 
     
     
       39. The method of claim 38 wherein said assaying is accomplished by atomic absorbtion spectroscopy. 
     
     
       40. The method of claim 37 wherein said ellagic acid or ellagic acid salt is sodium ellagate. 
     
     
       41. The method of claim 37 wherein the final molar concentration of ellagic acid or ellagic acid salt in the reagent is approximately 0.1 millimolar. 
     
     
       42. The method of claim 37 wherein said divalent metal ions are selected from the group consisting of: magnesium;   calcium;   copper;   cobalt;   iron;   lead;   manganese;   strontium; and,   zinc.   
     
     
       43. The method of claim 37 wherein the cephalin is soybean cephalin. 
     
     
       44. The method of claim 37 wherein said adjusting of the pH of said solution is accomplished by addition of a buffer. 
     
     
       45. The method of claim 44 wherein the buffer is N-2-hydroxyethylpiperazine-N 1  -2-ethanesulfonic acid hemisodium salt. 
     
     
       46. A method of making a stable reagent capable of forming a procoagulation reagent upon exposure to a source of cephalin comprising the sequential steps of: first, dissolving a known weight of an ellagic acid or an ellagic acid salt in an aqueous solution;   second, adjusting the concentration of divalent metal ions of said solution to achieve a divalent metal ion molar ratio of about 3 or less, but at least 0.1, relative to the ellagic acid or ellagic acid salt of said solution; and,   third, adjusting the pH of the resulting ellagic acid solution to approximately 7.5.   
     
     
       47. The method of claim 46 and further comprising assaying one or more of said ellagic acid or ellagic acid salt or said aqueous solution prior to said adjusting the divalent metal ion concentration. 
     
     
       48. The method of claim 47 wherein said assaying is accomplished by atomic absorbtion spectroscopy. 
     
     
       49. The method of claim 46 wherein said ellagic acid or ellagic acid salt is sodium ellagate. 
     
     
       50. The method of claim 46 wherein the final molar concentration of ellagic acid or ellagic acid salt in the reagent is approximately 0.1 millimolar. 
     
     
       51. The method of claim 46 wherein said divalent metal ions are selected from the group consisting of: magnesium;   calcium;   copper;   cobalt;   iron;   lead;   manganese;   strontium; and,   zinc.   
     
     
       52. The method of claim 46 wherein said adjusting of the pH of said solution is accomplished by addition of a buffer. 
     
     
       53. The method of claim 52 wherein the buffer is N-2-hydroxyethylpiperazine-N 1  -2-ethanesulfonic acid hemisodium salt.

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